Project Summary Our long-term goal is to elucidate the molecular mechanisms of dietary fat digestion by pancreatic lipases. In this application, we will focus on the steps in lipolysis that influence substrate specificity. Lipases differ from esterases that act on water soluble substrates in that lipases must absorb to the interface of the substrate emulsion before substrate binds in the active site. In addition, the archetype of pancreatic lipases, pancreatic triglyceride lipase (PTL), requires colipase to absorb to interfaces. Importantly, mutations in the absorption surface of colipase can change the substrate preference of the PTL-colipase complex underscoring the importance of absorption in determining substrate specificity. Thus, three distinct steps--colipase binding, interfacial absorption and substrate binding in the active site--all influence substrate specificity. To investigate the molecular elements that mediate the interaction of PTL with substrate, we will take advantage of a close PTL homologue, pancreatic lipase related protein 2 (PLRP2). These lipases have highly conserved primary structures, share a common three-dimensional structure and share common catalytic machinery; yet, they have quite different enzymatic properties. PTL prefers triglycerides whereas PLRP2 also cleaves galactolipids and phospholipids. Furthermore, PTL clearly requires colipase whereas the PLRP2 requirement for colipase remains controversial. Our central hypothesis is that specific structural domains govern each step of the interaction between pancreatic lipases and substrate. To identify domains involved in determining the substrate specificity of PTL and PLRP2, we will utilize available information on structure and kinetic properties of these lipases. We base this approach on multiple observations from our published and preliminary data. 1) Structural studies and our published studies of the PTL-colipase complex have guided the identification of distinct domains that influence colipase binding to PTL. 2) Analysis of structural data and our preliminary studies have identified critical residues in PTL-colipase and PLRP2 that influence the absorption of these lipases to lipid emulsions. 3) Comparison and preliminary analysis of the structures of PTL and PLRP2 identified domains that can mediate the differences in substrate specificity. Completion of our Specific Aims will provide molecular details about each step in the interaction of pancreatic lipases with substrate. Ultimately, we will be able to rationally engineer the enzymatic properties of lipases to meet specific therapeutic or industrial needs.